Polyprenyl phosphates are lipid-soluble vitamine-like substances which control (directly or indirectly) many physiological processes in all living organisms, from bacteria to human beings (see for example review Swiezewska E. and Danikiewicz W. Polyisoprenoids: structure, biosynthesis and function.//Progr. Lip. Res., 2005, v. 44, p. 235-258). From chemical point of view, they represent monoesters of phosphoric acid with linear long-chain isoprenoid alcohols—polyprenols belonging to isoprenoid class. In living cells, polyprenols and polyprenyl phosphates exist and function in the form of oligomer homologue families with the predominance of one or two compounds, and they are isolated in such a form from natural objects. Isolating polyprenyl phosphates from the natural sources is unprofitable owing to low content thereof, and complexity high labor intensity of the process. Therefore, the compounds are produced via chemical phosphorylation of plant polyprenols as a most available renewable raw material.
Polyprenyl phosphates are irreplaceable coenzymes of glycosyltransferases which participate in biosynthesis of carbohydrate-containing cell biopolymers; their content in biomembranes controls the rate of that process. Chemically synthesized polyprenyl phosphates were used and are used for elucidating in vitro mechanism of action of these compounds within living cells, mainly in regularities of their interaction with glycosyltransferase preparations isolated from the cells (see for example review Danilov L. L. and Druzhinina T. N. Chemical synthesis of dolichyl phosphates, their analogues and derivatives and application of these compounds in biochemical assays.//Acta Biochim Polon., 2007, v. 54, No 4, p. 695-701).
Recently, polyprenyl phosphates began to use for identifying cloned glycosyltransferases, which substrates those polyprenyl phosphates are, and for developing model biochemical in vitro test-systems which are useful for search new efficient antibiotics inhibiting those enzymes and blocking by a new principle a growth and propagation of pathogenic microorganisms which became resistant to existing pharmaceuticals (Borman S. Drug design: first glimpses of cell-wall-forming enzyme will aid search for new antibiotics.//Chem. Eng. News, 2007, March 12, p. 9).
From 80-th years of XX century the investigation of physiological action of exogenic (introduced from outside) polyprenyl phosphates was started and their physiological activity was demonstrated (EP 0149847, 1984, and EP 0165436, 1985). However, owing to unmanufacturability of methods for producing thereof, commercial drugs based on polyprenyl phosphates had not been created.
In 90-th years of XX century in Russian Federation, the remedy for prophylaxis and treatment of infectious diseases and correction of pathological conditions of living organism, the main active ingredients of which are polyprenyl phosphates, had been developed (RU 2129867, 1999, and U.S. Pat. No. 6,525,035, 2003).
For successful manufacture and realization of such preparations in sufficient amounts, a great value has the method for producing their active ingredients, which method must be simple, manufacturable and profit-proved.
Several methods for phosphorylation of polyprenols, non-universal, technically complicated or resulted in low yields, were described in literature before 1980 (see reviews Danilov L. L. and Shibaev V. N. Phosphopolyprenols and their glycosyl esters: chemical synthesis and biochemical application.//In: Studies in natural products chemistry (Atta-ur-Rahman ed., Elsevier, Amsterdam—Oxford—New York—Tokyo), 1991, v. 8, p. 63-114; Shibaev V. N. and Danilov L. L. Synthesis of intermediates in dolichol pathway of protein glycosylation.//In: Glycopeptides and related compounds: synthesis, analysis and applications (D. C. Large and C. D. Warren eds.), Marcel Dekker Inc., New York—Basel—Hong Kong, 1997, p. 427-504).
The first simple and efficient method of phosphorylation of polyisoprenoid alcohols with POCl3 (Danilov L. L., Chojnacki T. A simple procedure for preparing dolichyl monophosphates by the use of POCl3.//FEBS Lett., 1981, v. 131, p. 310-312) was proved to be applicable only for preparing 2,3-dihydropolyprenyl phosphates (dolichyl phosphates). On the basis of this method with small modifications, a method for producing phosphates of that polyprenol subclass was patented (EP 0149847, 1985; U.S. Pat. No. 4,792,615, 1986, and U.S. Pat. No. 5,306,714, 1994).
In 1988, a simple universal method for producing polyprenyl phosphates (including 2,3-dihydropolyprenyl phosphates) had been developed comprising a step of interacting C15-C95-polyprenols with substituted ammonium salts of phosphoric acid and trichloroacetonitrile in the medium of aprotic organic solvent at molar ratio polyprenol:phosphoric acid salt:trichloroacetonitrile of 1:(4-10):(25-50) followed by successive steps of treating the reaction product with ammonia, hydrolyzing with aqueous solution of 4-dimethylaminopyridine or N-methylimidazole, and isolating and purifying the target compounds by anion-exchange column chromatography (SU 1432065, 1988, taken as the closest analog, see Comparative Example 16 below). As a result, ammonium salts of polyprenyl phosphates were obtained.
Based at this principle of phosphorylation, similar procedures (with or without hydrolysis) for producing polyprenyl phosphates and their modified derivatives were described later (Danilov L. L., Maltsev S. D., and Shibaev V. N. Phosphorylation of polyprenols by tetra-n-butylammonium phosphate at the presence of trichloroacetonitrile.//Bioorgan. Khim, 1988, v. 14, No 9, p. 1287-1289; Danilov L. L. and Shibaev V. N. Phosphopolyprenols and their glycosyl esters: chemical synthesis and biochemical application.//In: Studies in natural products chemistry (Atta-ur-Rahman ed., Elsevier, Amsterdam—Oxford—New York—Tokyo), 1991, v. 8, p. 63-114).
Recently, a method for producing polyprenyl phosphates based on another principle, the interaction of polyprenols with pyrophosphoric acid in the presence of nitrogen base in the medium of aprotic organic solvent followed by basic hydrolysis of formed in the reaction process of symmetrical diester of pyrophosphoric acid (CN 1709894, 2008). The purification of obtained polyprenyl phosphates was achieved by column chromatography procedures, therefore, this method giving no advantages as compared with above method and its modifications.
The main drawbacks of above analogs are:
1) the non-optimized reagent ratio in carrying out the reaction;
2) in some cases, the necessity of hydrolysis for increasing yield of required compound;
3) the step of isolating and purifying the desired compounds by anion-exchange column chromatography;
4) using expensive import adsorbents such as DEAE-cellulose DE-52;
5) expending bulk of solvents for this purpose;
6) isolating of purified polyprenyl phosphates mainly in the form of ammonium salts unstable in storage and hardly solubilized in aqueous medium (the consequence of using ammonium acetate solutions as eluent at anion-exchange chromatography).
Ammonium salts of polyprenyl phosphates, which are convenient for producing and isolating in laboratory conditions in milligram quantities, were used and are often used up to the present for biochemical assays in cell-free enzymic systems (Danilov L. L. and Druzhinina T. N. Chemical synthesis of dolichyl phosphates, their analogues and derivatives and application of these compounds in biochemical assays.//Acta Biochim Polon., 2007, v. 54, No 4, p. 695-701). However, they are unsuitable for manufacturing medicinal agents in commercial scales and for investigating mechanism of physiological and therapeutic action of polyprenyl phosphates at cell and higher levels up to whole organisms, where pharmaceutically acceptable, stable and solubilizable in aqueous medium salts of polyprenyl phosphates are required. A cation nature and a substitution extent of phosphate group by those cations can substantionally impact on physico-chemical properties of the substances. Moreover, it is known that different cations themselves possess unequal physiological action on an organism.
So, the development of simple, manufacturable and profit-proved method for producing polyprenyl phosphates having a wide spectrum of physiologically accepted cations is important for creating new generation of pharmaceuticals on their basis, as well as for improving methods for biochemical investigations thereof.